Direct measurements of multiple adhesive alignments and unbinding trajectories between cadherin extracellular domains

Direct measurements of the interactions between antiparallel, oriented mono layers of the complete extracellular region of C-cadherin demonstrate that, rather than binding in a single unique orientation, the cadherins adhere i n three distinct alignments. The strongest adhesion is observed when the op posing extracellular fragments are completely interdigitated. A second adhe sive alignment forms when the interdigitated proteins separate by 70 +/- 10 Angstrom. A third complex forms at a bilayer separation commensurate with the approximate overlap of cadherin extracellular domains 1 and 2 (CEC1-2). The locations of the energy minima are independent of both the surface den sity of bound cadherin and the stiffness of the force transducer. Using sur face element integration, we show that two flat surfaces that interact thro ugh an oscillatory potential will exhibit discrete minima at the same locat ions in the force profile measured between hemicylinders covered with ident ical materials. The measured interaction profiles, therefore, reflect the r elative separations at which the antiparallel proteins adhere, and are unaf fected by the curvature of the underlying substrate. The successive formati on and rupture of multiple protein contacts during detachment can explain t he observed sluggish unbinding of cadherin monolayers. Velocity-distance pr ofiles, obtained by quantitative video analysis of the unbinding trajectory , exhibit three velocity regimes, the transitions between which coincide wi th the positions of the adhesive minima. These findings suggest that cadher ins undergo multiple stage unbinding, which may function to impede adhesive failure under force.